Whole blood is made up of many components, including red blood cells, white blood cells, platelets and plasma. Although whole blood is useful, e.g., in blood replacement therapy during surgical procedures, individual blood components also have substantial utility in replacement therapies, coagulation therapy, wound healing and tissue regeneration, as described below.
Red blood cells (RBCs) are flexible biconcave cells packed with hemoglobin which carry oxygen throughout the body. RBCs are the most dense whole blood component and thus can be readily separated from whole blood, e.g., by centrifugation or even by permitting settling under the force of gravity. Packed red cells can be transfused back into a donor patient (autologous transfusion) or transfused into a patient with a compatible blood type, e.g., having an appropriate A, B, AB, O and rh antigen type.
White blood cells (WBCs) are a diverse array of cell types, e.g., lymphocytes, macrophages, and polymorphonuclear neutrophils (PMNs), which are primarily involved with immune responses and fighting infections. WBCs are generally somewhat less dense than RBCs and, together with platelets, form a white “buffy coat” layer on top of RBCs during centrifugation of whole blood. Buffy coats can be a useful source of growth factors, blast cells and cytokines.
Platelets are cell fragments shed into the blood stream by large megakaryocyte cells in the bone marrow. Platelets play an important role in coagulation of blood. When platelets contact damaged blood vessels, they aggregate and send chemical messages that can initiate the coagulation cascade. Patients with reduced peripheral blood platelet counts, e.g., patents with certain leukemias or sickle cell anemia, may require transfusions of platelets to help control bleeding episodes. Platelets settle through plasma more slowly than RBCs and WBCs due to their low density and small size. When whole blood is centrifuged, platelets can be found distributed in the plasma, in the buffy coat, and/or in the upper portion of the red cells, depending on the centrifugation time and centripetal forces involved.
Plasma is a complex aqueous solution of proteins, lipids, small molecules and salts which acts as the transport medium for the other blood components. Plasma also contains elements necessary to the function of various biological systems, e.g., the coagulation cascade, the compliment cascade, hormones, buffers, nutrients, etc. Plasma can be stabilized with anticoagulants, e.g., citrate or heparin, for handling or storage. Plasma is the least dense of the blood components described here and presents as supernatant after centrifugation of whole blood.
Plasma and/or platelets, in the absence of active anticoagulants, or in the presence of a catalyst, e.g. thrombin, can initiate coagulation or clotting to form a wound sealant or platelet gel useful to repair damaged tissues and to stop bleeding. Such wound sealants are generally more effective when they are concentrated and contain abundant platelets. Fibrin based wound sealants produced from pooled blood components have been available in Europe. Licensing of such products for use in the United States has been prolonged due to fear of disease transmission, though the first plasma-based fibrin sealant from pooled allogeneic sources was recently cleared. However, wound sealant preparations made from a patient's own (autologous) blood present minimal risk of disease transmission.
Plasma can be harvested by filtration of blood directly from a patient. For example, in U.S. Pat. No. 4,498,983 to Bilsted, “Pressure Cuff Draw Mode Enhancement System and Method for a Single Needle Blood Fractionation System”, whole blood is drawn through a needle into an apparatus wherein the blood is treated with an anticoagulant and passed over a filter to remove some plasma. The plasma depleted blood is then reinfused into the patient along with some saline to make up the blood volume. Such filtered plasma will form a clot when exposed to thrombin. However, the concentration of fibrinogen in filtered plasma is too low to act as an effective wound sealant.
A plasma preparation can also be produced by centrifugation of whole blood (see, e.g., U.S. Pat. No. 3,145,713, “Method and Apparatus for Processing Blood” and U.S. Pat. No. 4,151,844, “Method and Apparatus for Separating Whole Blood into Its Components and for Automatically Collecting One Component”). Some platelets may remain in centrifuged plasma of these inventions to add some structure when it is clotted. However, without concentration, such plasma does not have the properties of an adequate fibrin glue.
A fibrin glue wound sealant can be prepared by cryoprecipitation and centrifugation of plasma to concentrate fibrinogen. For example, a fibrin glue can be prepared by holding anticoagulated plasma in the cold to precipitate coagulation proteins, e.g., fibrinogen, followed by centrifugation to pellet and concentrate the cryoprecipitate. Just before application to a wound, thrombin is added to the cryoprecipitate to begin conversion of fibrinogen to fibrin whereby the fibrin glue clot is formed. The fibrin glue can stabilize surgical incisions and seal leaky blood vessels. Although such procedures can be helpful, they suffer from the time and effort required, the waste of disposed blood components, and the bulky equipment involved. Cryoprecipitated fibrin glues, particularly from autologous plasma, are not a practical solution to wound sealing on an emergency basis.
In U.S. Pat. No. 5,585,007 to Antanavich, “Plasma Concentrate and Tissue Sealant Methods and Apparatuses for Making Concentrated Plasma and/or Tissue Sealant”, whole blood is separated by centripetal force and the plasma is concentrated by selective absorption of water. This device is an improvement over prior technology in that it both separates and concentrates plasma. Fifty (50) ml of whole blood is introduced into the top centrifugal chamber of the device, then the chamber is spun to sediment blood cells about the periphery of the chamber where they are captured, e.g., by a mat matrix. The rotation of the chamber is stopped, allowing liquid components of the blood to fall into a lower chamber where they are exposed to dehydrating beads which concentrate the liquid components to some extent.
The Antanavich device lacks the flexibility required to accommodate different amounts of whole blood starting material, to adjust for variations in blood component proportions, or to control the character of concentrated products. For example, depending on the hematocrit of the whole blood, various amounts of liquid components remain trapped in the upper chamber and various proportions of water are removed by the dehydrating beads. Fine tuning the device to harvest special component mixtures, e.g., buffy coat, is not possible. In addition, many blood components, e.g. white blood cells, can not be collected or reinfused as they are lost in the device as waste material.
There remains a need to provide an automated system to quickly and consistently separate and concentrate selected blood components in a single aseptically sealed instrument. Waste of blood components needs to be reduced by provision of technologies for high recovery and reconstitution of unselected blood components for reinfusion.